A functional host immune system consists of different host components (e.g. effector cells and molecules) that act coordinately to recognize microbial infections, dying, and malignant tissue and elicit the appropriate responses, respectively. The immune system is divided into 2 broad categories, the innate and adaptive immune responses. The innate immune response consists of a variety of cellular and molecular effectors such as dendritic cells, macrophages, eosinophils, neutrophils, and natural killer cells. The adaptive immune response consists of T lymphocytes, B lymphocytes, and natural killer T (NKT) cells. The innate and adaptive are intimately linked and often work jointly to maintain the overall health of the individual. T lymphocytes (T cells) refers to a family of both effector and regulatory cells. The helper (CD4 Th) T cells and the cytotoxic (CD8, CTL) T cells are the major effector T cells. CD4 Th cells support the development of an adaptive immune response by producing cytokines for B cells and CD8 T cells. CD4 T cells can be further subdivided into Th1, Th2, and Th17 cells, each with a specialized function in the immune response.
Adaptive and innate immune responses are regulated by regulatory T cells and NKT cells. The adaptive immune response is activated by the innate immune system and particularly macrophages and dendritic cells which have taken up antigen derived from dysfunctional or infected tissues. An inflammatory response refers to an active immune response that is mediated by one or more immune effectors of the innate and/or adaptive immune system.
Lipopolysaccharide (LPS), often called endotoxin as it is isolated from E. coli and other gram-negative bacteria, is a well known and potent inflammatory immune activator. Ulevitch and Tobias, Curr. Opin. Immunol. 11: 19-22 (1999). Several studies have validated the important role of LPS in triggering inflammation in response to bacterial infection. Although the chemical structure of LPS has been known for some time, the molecular basis of recognition of LPS by serum proteins and/or cells is only now being elucidated.
A family of receptors, referred to as Toll-like receptors, (TLRs), have been linked to LPS and other microbial components to activation of the adaptive and innate immune responses. TLRs are membrane proteins having a single transmembrane domain, a cytoplasmic domain that shares similarity with the cytoplasmic domain of the IL-1 receptor, and a relatively large extracellular domain that may contain multiple ligand-binding sites. The importance of TLRs in the immune response to LPS has been demonstrated for at least two TLRs, TLR2 and TLR4.
In addition to TLRs, at least two other proteins are part of the host innate defense pathway that responds to bacterial LPS. The lipopolysaccharide binding protein (LBP) is an acute-phase serum protein that binds LPS molecules. The LBP then facilitates the transfer of LPS to CD14, in its membrane-bound form as mCD14, and/or its soluble form found in serum, sCD14. The importance of CD14 in inflammation has been validated in mice, where CD14 was shown to be required for the development of sepsis.
One role of CD14 is to concentrate microbial components such as LPS on the host cell surface for further recognition by TLRs and the innate host response system. CD14 acts with TLRs as a co-receptor to facilitate activation of host cells. Transfer of E. coli LPS by either mCD14 or sCD14 to a cell-associated TLR4 and MD-2 protein complex has been shown to initiate host cell activation pathways leading to innate host defense mediator production. Muta and Takeshige, Eur. J. Biochem. 268:4580-4589 (2001); da Silva Correia et al., J. Biol. Chem. 276: 21129-21135 (2001).
The biologically active endotoxic moiety of LPS is lipid A, a phosphorylated, fatty acid-acylated glucosamine disaccharide that serves to anchor the entire LPS structure in the outer membrane of Gram-negative bacteria. Host responses to LPS vary significantly depending upon lipid A structure. Biological activity can be affected by the number, chain length, and position of lipid A fatty acids as well as the number of phosphate groups attached to the glucosamine disaccharide backbone. Takada and Kotani, 1992, Bacterial Endotoxic Lipopolysaccharides, Boca Raton: CRC Press; Loppnow et al., J. Immunol. 142: 3229-3238 (1989); and Schumann et al., Blood 87: 2805-2814 (1996).
Many efforts have been made to obtain nontoxic lipid A while retaining the beneficial activities. The toxic effects of lipid A can be ameliorated by selective chemical modification of lipid A to produce monophosphoryl lipid A compounds. Methods of making and using these compounds as immunostimulants, vaccine adjuvants, and as monotherapies, have been described. See, e.g., U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034; 4,912,094; 4,987,237; 6,491,919; 6,800,613; and 6,911,434.
Porphyromonas gingivalis is a Gram-negative bacterium that is an important etiologic agent of adult type periodontitis. It releases large amounts of vesicles containing LPS that penetrate periodontal tissue and participate in a destructive innate host response associated with the disease. LPS and its lipid A component from P. gingivalis, however, do not elicit host responses in a manner similar to the classic E. coli endotoxin. P. gingivalis LPS is both less potent and it elicits a different pattern of inflammatory mediators when compared to E. coli LPS. For example, P. gingivalis LPS is not as potent an activator of human monocytes as E. coli LPS. Further, some P. gingivalis LPS fractions, unlike E. coli LPS, do not cause E-selectin expression on human endothelial cells, and some P. gingivalis LPS fractions are a natural antagonist of human endothelial cells and the IL-8 response to E. coli and other oral bacteria. Bainbridge, B. W. and Darveau, R. P., 1997, in Morrison, D., ed., Endotoxin in Health and Disease, New York, Marcel Dekker, p. 899-913. Based at least in part on differences in biological activities between E. coli and P. gingivalis LPS, the use of preparations of P. gingivalis LPS and lipid A and derivatives thereof to elicit a Th2 immune response has been proposed in U.S. Pat. No. 6,818,221 of Pulendran et al.
One structural study of the lipid A found in P. gingivalis LPS preparations showed that the major lipid A species is a tri-acylated monophosphorylated form with a negative ion mass of 1195. Ogawa, FEBS Lett. 332:197-201 (1993); see also, U.S. Pat. No. 5,654,289 to Kodama et al. In another study, multiple P. gingivalis lipid A structural isoforms were observed, however two forms predominated which were tetra-acylated monophosphorylated forms with molecular mass ions of 1435 and 1449, respectively. Kumada et al., J. Bacteriol. 177:2098-2106 (1995). These structures and other isoforms found at m/z 1770 and 1690 are depicted in FIG. 1, and differ from the canonical E. coli lipid A structure in the number of phosphates, and the number, type, and position of the fatty acid chains. Another study employing tri-reagent to extract LPS has found that P. gingivalis contains multiple lipid A species. Yi and Hackett, Analyst 125:651-656 (2000).
Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject to which the antigen is administered, or enhance certain activities of cells from the immune system. An adjuvant admixed or administered with, or during the course of antigenic stimulation is typically referred to as a vaccine or vaccination. The adjuvant provides the immune alerting or “danger” signal. A number of compounds exhibiting varying degrees of adjuvant activity have been prepared and tested. However, these and other prior adjuvant systems often display toxic properties, are unstable and/or have unacceptably low immunostimulatory effects.
An adjuvant presently licensed for human use in the United States is alum, a group of aluminum salts (e.g., aluminum hydroxide, aluminum phosphate) in which vaccine antigens are formulated. Particulate carriers like alum reportedly promote the uptake, processing and presentation of soluble antigens by macrophages. Alum, however, is not without side-effects and is unfortunately limited to humoral (antibody) immunity only.
The discovery and development of effective adjuvant systems is essential for improving the efficacy and safety of existing and future vaccines and other immunotherapies. Thus, there is a continual need for new and improved adjuvant and immunomodulator systems to better facilitate the development of a next generation of vaccines and immunotherapies. The present invention addresses these and other related needs.